Fluorescence polarization based displacement assay for the determination of small molecules with aptamers.
The conversion of an aptamer-target binding event into a detectable signal is an important step in the development of aptamer-based sensors. In this work, we show that the displacement of a fluorescently labeled oligo from the aptamer by the target can be detected by fluorescence polarization (FP). We used Ochratoxin A (OTA), a small organic molecule (MW = 403) as a case study. A detection limit of 5 nM OTA was achieved. The method presented here provides an advantage over fluorophore-quenching systems and other steady-state fluorescence approaches in that no modification of the aptamer or the target is required. Additionally, the signal is produced by the displacement event itself, so no further aggregation or conformational events have to be considered. This analytical method is particularly useful for small targets, as for large targets a direct measurement of the FP change of a labeled aptamer upon binding can be used to determine the concentration of the target. The results presented here demonstrate that aptamers and inexpensive labeled oligos can be used for rapid, sensitive, and specific determination of small molecules by means of FP.
Cruz-Aguado
Jorge A
JA
NeoVentures Biotechnology Inc., 700 Collip Circle, London, Ontario, N6G 4X8, Canada.
Penner
Gregory
G
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D009793:Ochratoxins / Q000032:analysis* / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics
D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D009793:Ochratoxins; D009838:Oligodeoxyribonucleotides; C025589:ochratoxin A